For our first specific aim, we have created a series of transgenic mice which either overexpress methionine sulfoxide reductase in specific cellular locations or which lack the enzyme. We have established cell cultures of embryonic fibroblasts from these animals and have challenged them with a series of oxidative stresses and compared their response to that of wild-type fibroblasts. In two collaborative studies with other NIH investigators, we are studying the susceptibility of the transgenic heart to ischemia and reperfusion and of the eye to light-induced oxidative damage. We have also delineated the mechanism by which a single mammalian gene is able to encode methionine sulfoxide reductase directed to both the cytosol and mitochondria. Work on the second specific aim has progressed rapidly after the development of very sensitive assays for iron regulatory protein-2. We confirmed the published observation that iron-deficient cells suddenly exposed to high concentrations of iron rapidly degrade iron-regulatory protein-2 through the proteasome pathway. However, when examining the physiological regulation of cells not stressed by exposure to high concentrations of iron, we found that turnover of the protein is not mediated by the proteasome. We have shown that the proteolytic system responsible for the non-proteasomal turnover is calcium-dependent but is not a calpain. We have employed modern analytical techniques coupled with classical biochemical fractionation to identify compounds within Deinococcus radiodurans which are radioprotective. Those identified thus far are the metal manganese, a mixture of bases and nucleosides, and a high concentration of low molecular weight peptides. Synthetic mixtures are highly protective of proteins, including functional activity of enzymes.